Calpains are family of calcium-activated neutral proteases involved with signal transduction and cell motility, but whose overactivation is strongly implicated in neurodegeneration following traumatic brain injury and other CMS insults and disorders. Although studies over the past 30 years have provided a wealth of knowledge regarding calpains, important questions remain: Which calpain isoforms are responsible for the neurodegeneration? How is calpain activated postinjury? What are the critical substrates? Are m- and u- calpain located in the same subcellular compartment? We recently found that mitochondria contain u-calpain (calpain 1 plus calpain small subunit 1). Based on the existing data and our preliminary results, our working model is that mitochondrial u-calpain is situated within the intermembrane space, is activated following opening of the mitochondrial permeability transition pore (mPTP), and that mitochondrial substrates of u- calpain include apoptosis inducing factor (AIF), which when cleaved by calpain is released into the cytosol and translocates to the nucleus to contribute to caspase-independent cell death. In addition, we propose that activated u-calpain is released from the mitochondria into the cytosol, where its substrates are similar to those of m-calpain. In summary, mitochondrial u-calpain is hypothesized to become activated following mPTP opening and function as a death-related protease. We therefore hypothesize that neurons deficient in calpain 1will exhibit resistance to excitotoxic death and that neurodegeneration and functional impairment following traumatic brain injury will be markedly attenuated in mice deficient in u-calpain. Interactions with other projects include evaluating the ability of calpastatin (Project 1) and small molecule calpain inhibitors (Project 2) to inhibit mitochondrial u-calpain. This project will use the same injury models, methods of assessing functional impairment, and methods for evaluating calpain activity as other projects, enabling direct comparison of results. In addition, we will collaborate with the Proteomics and Biomarker Core C to identify novel substrates of mitochondrial u-calpain. Together the experiments outlined in this project will enhance our understanding of the function of the u-calpain isoform, mechanisms involved in its activation, and its role in neuron death following traumatic brain injury and other CNS insults.